Since the end of 1987, oligonucleotide-directed triple helix formation (Le Doan et al, 1987; Moser et al, 1987) has risen considerable interests because it provides an elegant rational basis for gene-specific DNA targeting, therefore for interfering with gene expression at transcriptional level and other biological process (Hélène et al, 1991; Chan et al, 1997). In the last decade, intense research in this field has paved physico-chemical and biochemical basis for developing high affinity and nuclease-resistant triple helix-forming oligonucleotides (TFOs) (Thuong et al, 1993; Sun et al, 1996). It has been shown that transcription can be effectively inhibited by forming stable triple helix that competes either with the binding of transcriptional factors at gene regulation region (promoter/enhancer) or with the elongation of RNA polymerase (Guieysse et al, 1996; Giovannangeli et al, 1996). Furthermore, it has been demonstrated that, at least, some of the target DNA sequences in chromatin environment are accessible to TFOs (Gionvannangeli et al, 1997; Belousov et al, 1998).
In parallel with the development of the so-called “antigene strategy”, sequence-specific targeting of Watson-Crick double-stranded DNA (dsDNA) has been exploited to create diverse new tools for molecular and cellular biology (Maher et al, 1996; Thuong et al, 1993). TFOs have been successfully converted to artificial nucleases by covalent attachment of a DNA cleaving reagent (including metal chelating complexes, alkylating or photoactivating agents and nucleases) (Moser et al, 1987; Francois et al, 1989; Pei et al, 1991) or by combination of a couple of methylase/restriction enzyme (known as Achilles Heel approach) (Strobel et al, 1991) or most recently by recruiting topoisomerase (Matteucci et al, 1997; Arimondo et al, 1999). It has also been used to bend DNA (Akiyama et al, 1996) and to padlock dsDNA (Escudé et al, 1999).
Inter-strand cross-linked DNA by TFO-psoralen conjugates has been shown to trigger DNA repair events that lead to site-specific mutagenesis (Havre et al, 1993; Barre et al, 1999). Such an application of TFOs is potentially useful because many known disease and pathologies originate from mutation in DNA sequences. In general, the correct repair of mutant gene either by directed mutagenesis or by homologous recombination might help to cure these diseases. However, the lack of efficient tools for directed mutagenesis in mammal cells has so far hampered significant progress in this field in part due to very low frequency of homologous recombination (10-8-10-5) (Bollag et al, 1989) and high background of random integration of transfected DNA (Roth et al, 1986).
Recent progress has been made in targeted gene repair directed by the chimeric RNA/DNA oligonucleotides in mammalian cells (Yoon et al, 1996; Alexeev et al, 1998 and Cole-Strauss et al, 1999).
The current development of triplex-directed mutagenesis and recombination benefits from the sequence-specific recognition of dsDNA by TFO and the efficient DNA inter-strand cross-linking by a mutagen, namely psoralen which is covalently attached to a TFO.
WO 96/41 008 discloses a method for effecting homologous recombination between a native nucleic acid segment and a donor nucleic acid by formation of a triple helix between an oligonucleotide and one or two DNA strands in the vicinity of a target region wherein recombination is to occur, the oligonucleotide being capable of triggering an homologous recombination in the target region with the donor nucleic acid segment.
The triple helix-forming oligonucleotide is chemically modified to include a mutagen at either the 5′ end, 3′ end, or internal portion so that the mutagen is proximal to a site where it will cause modification or damage to the nucleic acid.
However, mutational event generated by error-prone repair of such a mutagen-TFO conjugate generally produces an unpredictable and a widespread spectrum of mutations and deletions. Therefore, its potential use as a therapeutic agent is compromised, especially when one think about the well known genotoxic and mutagenic effect of mutagenesis themselves.
Chan et al (1999) disclose a method of homologous recombination based on oligonucleotid-directed triple helix formation, wherein the TFO segment is covalently attached via a flexible linker to the donor segment.
Culver et al (1999) disclose a sequence-specific genomic targeting system for the correction of chromosomal mutations comprising two different binding domains incorporated into a single-stranded nucleotide. Namely, the targeting system comprises a first domain capable of forming a triplex by means of Hoogsteen interactions and a second domain capable of forming a heteroduplex which is covalently attached to the first domain by means of a linker segment.
However, there was a need for a new method of directed mutagenesis based on oligonucleotide-directed triple helix formation and homologous recombination which has a high efficiency of recombination, is mutagen-free, and which allows mutagenesis at a site which is not distance-limited on the triple helix-forming site.